Accumulating data suggest that assembly and budding of different viruses, including HIV-1, takes place in glycosphingolipid and cholesterol enriched lipid rafts. Although a direct involvement of rafts in HIV-1 entry has not yet been presented, recent studies strongly suggest this possibility. Our preliminary results demonstrate that that HIV-1 entry into CD4+ T cells requires the presence of intact lipid rafts. This possibility was further substantiated by the observation that HIV-1 receptors, CD4, CXCR4, and CCR5, associate with lipid rafts. Since HIV-1 entry requires cooperation between multiple virus receptors, distribution of these receptors in different rafts may have important implications for the rate and efficiency of membrane fusion and HIV-1 entry. We hypothesize that effective raft aggregation is required to concentrate receptors locally to levels necessary for virus fusion and entry. Furthermore, we hypothesize that HIV-1 stimulates raft aggregation by inducing signaling upon binding to CD4 and/or coreceptors. In this proposal we will address the following issues: in Specific Aim number 1, we will determine whether the association of HIV-1 receptors with lipid rafts and the integrity of rafts is required for HIV-1 entry into CD4+ T lymphocytes. This will be achieved by analyzing how modification or disruption of rafts affects virus entry. In Specific Aim number 2, we will investigate the distribution of HIV-1 receptors in lipid rafts. We hypothesize that receptors used by HIV-1 may be distributed non-randomly in different raft populations. In Specific Aim number 3, we will elucidate the molecular mechanism(s) targeting CD4 receptors into lipid rafts. We believe that HIV-1 entry may be inhibited by preventing localization of the CD4 receptors to lipid rafts. Finally, in Specific Aim number 4, we will examine the molecular mechanism(s) of HIV-1-induced raft aggregation paying special attention to the role of HIV-1-induced signaling in this process. We believe that this study will provide a rational basis for a new therapeutic approach to interfere with HIV-1 entry.